The cytotoxicity of oxaziridines photogenerated after irradiation of chlordiazepoxide (CDZ) and its metabolites was investigated in vitro by a MTT assay on P388 leukemia and B16 melanoma cell lines and compared with that of the anticancer drug, melphalan. For the longer time-exposure experiment, oxaziridines had the same cytotoxicity as melphalan and this toxicity was higher when oxaziridines were photogenerated in the presence of cells. In conclusion, oxaziridines generated after CDZ, demoxepam, and desmethylchlordiazepoxide ultraviolet irradiation exhibited cytotoxicity activity against cancer cell lines. A possibility of CDZ use within the context of photodynamic therapy as a treatment for small, superficial tumors should not be excluded, because oxaziridines can be generated locally by skin-tumor local irradiation after CDZ topical administration.